c-Src-mediated phosphorylation of AP-2 reveals a general mechanism for receptors internalizing through the clathrin pathway.
Clathrin-mediated endocytosis is a complex process regulated at many different levels. We showed previously that activation of the angiotensin type 1 receptor (AT1R), which belongs to the G protein-coupled receptor (GPCR) family, leads to c-Src-dependent tyrosine phosphorylation of beta2-adaptin, a subunit of the clathrin adaptor AP-2. The phosphorylation of beta2-adaptin on tyrosine residue 737 (Y737) negatively regulates its interaction with betaarrestin, another important clathrin adaptor for GPCR internalization. Here we sought to determine whether AP-2 phosphorylation represents a general mechanism for different receptors internalizing through the clathrin pathway. Using a specifically designed antibody against the phosphorylated form of Y737 on beta2-adaptin, we demonstrate that this residue is phosphorylated by AT1R in different cell types like HEK293, COS-7 and vascular smooth muscle cells. Using RNA interference approaches, we reveal that this agonist-mediated event is both betaarrestin- and c-Src-dependent, and that it occurs at the plasma membrane in clathrin-coated vesicles (CCVs). We further show that this is not only a common event employed by other GPCRs like the beta2-adrenergic, vasopressin V2, bradykinin type 2, platelet-activating factor and endothelin A receptors but that the epidermal growth factor receptor is capable of eliciting the phosphorylation of AP-2 in CCVs. Our results imply that tyrosine phosphorylation of Y737 on beta2-adaptin is a common regulatory mechanism employed by different receptors undergoing clathrin-dependent endocytosis, and suggest a wider function for this event than originally anticipated.
Zimmerman
Brandon
B
Hormones and Cancer Research Unit, Department of Medicine, McGill University Health Center Research Institute, Royal Victoria Hospital, 687 Pine Avenue West, Montréal, Quebec, Canada H3A 1A1.
Simaan
May
M
Lee
Mi-Hye
MH
Luttrell
Louis M
LM
Laporte
Stéphane A
SA
D033962:Adaptor Protein Complex 2 / Q000378:metabolism*; D033966:Adaptor Protein Complex beta Subunits / Q000819:agonists / Q000378:metabolism; D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D019390:Arrestins / Q000378:metabolism; D019556:COS Cells; D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D004705:Endocytosis; D066246:ErbB Receptors / Q000378:metabolism; D019085:Fluorescent Antibody Technique, Direct; D055785:Gene Knockdown Techniques; D006801:Humans; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D044140:Receptor, Angiotensin, Type 1 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D019061:src-Family Kinases
D033962:Adaptor Protein Complex 2; D033966:Adaptor Protein Complex beta Subunits; D019390:Arrestins; D002966:Clathrin; D044140:Receptor, Angiotensin, Type 1; D043562:Receptors, G-Protein-Coupled; D000804:Angiotensin II; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human